Test chamber for growth and metabolism studies of biological cells



Aug. 6, 1968 L. A. IRVINE TEST CHAMBER FOR GROWTH AND METABOLISM STUDIES OF BIOLOGICAL CELLS Filed April 20, 1965 2 Sheets-Sheet l 5 6 m a M M E e W Z mm 64M. M a 0 mm \b 3 p mm 5 m Aug. 6, 1968 A IRVINE 3,395,034

TEST CHAMBER FOR GROWTH AND METABOLISM STUDIES OF BIOLOGICAL CELLS Filed April 20, 1965 2 Sheets-Sheet 2 I m my N WMWQNM m a? M W W m United States Patent 3,396,084 TEST CHAMBER FOR GROWTH AND METAB- OLISM STUDIES OF BIOLOGICAL CELLS Laurence A. Irvine, Takoma Park, Md., assignor t0 the United States of America as represented by the Secretary 0f the Air Force Filed Apr. 20, 1965, Ser. No. 449,656 4 Claims. (Cl. 195-127) The invention described herein may be manufactured and used by or for the United States Government for governmental purposes without the payment to me of any royalty thereon.

This invention relates to a device for monitoring the cellular growth and metabolism of cells. More specifically it relates to the disclosure of the construction of a hermetically sealed biocell having a controlled test chamber. This biocell is ideally suited for use in space satellites to measure various parameters of environmental conditions, such as zero gravity on the growth of biological cells. The control over the test chamber provides for maintaining the separation of two solutions for an extended period of time, as might be encountered in a delayed countdown at launching then at a suitable time on the command from an electrical signal a barrier between the solutions is removed allowing the solutions to mix and thus enabling the metabolism process to commence. The transmission characteristics of light travelling through the chamber are used as a means to indicate the removal of the barrier and as a means of monitoring any changes that take place in the material contained in the cell chamber.

The barrier separating the solutions is a stretched, rupturably elastic membrane. It is ruptured by the gas pressure built up by the electrolysis of one solution forcing it against a needle point. When the membrane is forced into contact with the needle point it ruptures and the inherent elasticity of the material accelerates the spread of the rupture until essentially the total barrier is removed. The electrolysis is initiated by an electrical signal injected into the solution by electrodes. This electrical signal may be energized on demand by an operator, it may be programmed with other events, or it may be initiated on a timed basis. The gases evolved during the electrolysis process being confined with the solution, will readily redissolve and produce no undesirable denaturation or other ill effects on the medium.

In order to conserve control elements and signals, the electrical signal initiating the electrolysis also supplies the energy to light the lamp and activate the electrical bridge used in the monitoring of the solution. The electrolysis is interrupted and terminated by a series fuse in the electrolysis circuit opening at a predetermined value of current flow. A wide selection of fusing may be used, including slow-blow varieties, to provide proper electrolysis time regardless of the strength of the electrolytic solution. It is obvious that the gas pressure built up by the electrolysis of the solution is dependent on the magni tude of the current flowing in the electrolyte, the duration time of the flow, and the electrolytic characteristics of the solution. The operation of the biocell device has been satisfactory even with very weak electrolytic solutions.

A bridge circuit, with two photoconductive units commonly called photocells, in two arms of the bridge, may be used to furnish the electrical signal output by which the biocell is monitored. The biocell is constructed such that light emanating from a small bulb activates the photocells. The chamber of the biocell containing the biological material is disposed between the lamp and one photocell, requiring the light from the lamp to traverse the chamber and its contents before reaching the photocell. The lamp shines directly on the second photocell. By using a bridge 3,396,084 Patented Aug. 6, 1968 circuit in this way variations in the illumination from the bulb which might otherwise tend to indicate biological changes are minimized. As the culture in the biocell changes its light transmission characteristics change, causing the balance of the electrical bridge to change thus providing a change in the bridge output potential. Generally as cell metabolism increases the transparency of the solution decreases. In many instances it is desirable to first calibrate the electrical output of the cell versus known culture concentrations of a specific biological specimen under known or standard environmental conditions before sending the equivalent biological material in the biocell to other environments. I

It is an object of the present invention to provide a biological test chamber that may be remotely activated and monitored.

It is another object of the present invention to provide a hermetically sealed biological test chamber that may be electrically activated and monitored with only one electrical input and one electrical output.

It is another object of the present invention to provide a biological test chamber that may be activated on command and monitored while the test chamber is experiencing conditions of zero gravity.

These and other objects will become apparent to those skilled in the art as the invention is better understood by the comprehension of the following description and drawings in which:

FIG. 1 is a simplified pictorial representation of the biocell;

FIG. 2 is an exploded view of the biocell showing detailed construction features; and

FIG. 3 is a schematic diagram of the electrical circuit of the biocell.

Refer to FIG. 1 which is an assembled view of the biocell less the electrical wiring and bridge resistors and to FIG. 2 which is an exploded view of the same cell showing mechanical construction details. The body of the cell is assembled from three sections 1, 2 and 3 of light permeable material such as methacrylate. The cell is held together in compression by the machine screws 4, 5, 6, 7 and 8. Clearance for the screws is provided in sections 1 and 2 with section 3 being tapped to receive the screw threads. Metal cover 9 encloses wiring cavity 10 and supports the connecting plug 11. It is provided with an access hole 12 for fuse and connector assembly 13 so that fuses may be changed without disassembling the cell. The plate is held in place with four screws 14, 15, 16 and 17.

The center section 2 contains the chamber 18 which is the cell proper. The center section also contains the small filling port 19. This port provides a means of inducing the solution into the cell chamber after assembly and thus precludes the accidental rupture o fthe membrane 20 by mechanically induced pressure on the membrane during assembly. The small well 21 over which the rupturable elastic membrane 20 is stretched and held in place by ring 22 is used to store the inoculum of resting cells. Membrane 20 may be fabricated of thin rubber material that is impervious to biological solutions. Stainless steel has been found to be a suitable material from which to construct ring 22. The stainless steel needle 23 is seated and partially embedded in the recess 24 of section 1. It is positioned such that the point of the needle 63 is in proximity to, but spaced apart from the face of the diaphragm 20 when the diaphragm is stretched and in place over the Well 21.

The O-rings 25 and 26 are compressed into their respective grooves when the cell is assembled and seal the chamber 18. Filling port 19 is sealed with stainless steel screw 27 and compression gasket 28. Photocell 29 is positioned in recess 30 to receive light from light emitting electric light bulb 31 that has traversed the chamber and its contents. Another similar photocell 32 is postioned in shaft 33. Its exact position in the shaft is not critical. It is important that it be cemented or otherwise held fixed in position. Wire leads 34 from the photocell 32 are fed through shaft 35 to electrical connecting cavity 10. Bulb 31 is positioned in shaft 36. Its light penetrates the thin wall section 37 of the light permeable material of which the biocell is fabricated and on into the specimen growing chamber 18. The light from the bulb also travels through a small portion of the shaft 33 and falls unobstructed on the photocell 32.

Electrodes 38 and 39 extend through the material of section 3 into the cell chamber 18. They are pressed through and sealed, if necessary, to the material of section 3 so as to maintain the hermetically sealed integrity of the test chamber. Fuse holder 40 with electrical connecting lugs 41 and 42 is positioned in slotted recess 43. The recess for the fuse does not extend through the section. All electrical connections and the electrical resistors for the bridge circuit are made within the cavity 10. The electrical circuit for the photocell 29 is brought to cavity 10 through electrical connectors 44 through 51, and the photocell conductors 52 and 53. The connectors may also serve as guide pins for aligning the sections.

FIG. 3 schematically sets forth the electrical circuits of the biocell. In a specific operating embodiment of the invention the photocell 29, which senses the light traversing biocell chamber 18 and the photocell 32 which senses directly the light output from the lamp 31, are Clairex type CL-404 photocells having a light resistance of 1500 ohms. The lamp 31 is a Chicago miniature lamp type #327, and the resistors 54 and 55 are 2,500 ohms, A1 watt. Electric energy source 58 is a twenty-eight volt supply. Control signal line 60 actuates the energy source to turn on and activate the biocell. It may be a control signal from a radio receiver. When chamber 18 is clear slightly more light reaches photocell 32 than photocell 29; thus in this embodiment the electrical bridge is never perfectly balanced and biocell output line 57 will always be positive with respect to line 56. As the culture thickens in the test chamber more and more light is cut off from reaching photocell 39 and the output voltage increases. Utilization device 59 may be a subcarrier oscillator with output 61 fed into a telemetry system. In some systems it may be desirable to include a Zener diode 62 across the output lines to limit the magnitude of voltage peaks entering the utilization system.

To further comprehend the operation and use of the biocell a typical preparation will be detailed: The unit is disassembled in a sterile glove box and all parts are exposed to ethylene oxide gas for sterilizing purposes. After sterilization, the inoculum, consisting of aqueous suspensions of resting cells, lyophilized cells, bacterial spores or other biological cells are placed in the inoculum well. The well is covered by the thinly stretched rubber membrane held in place by slipping the stainless steel ring over the membrane and well. The biocell is then assembled and the aqueous medium introduced through the filling port of the test chamber after which the port is sealed with the stainless steel screw and O-ring compression gasket. At this point the biocell containing the inoculum and medium, may be held for some period of time before the actual initiation of mixing and growth.

To initiate the mixing of the cells and the media the voltage is applied to the electrode pins. Electrolysis of the solution takes place due to the current flow and gases are evolved. Since the gases are confined by the chamber and the elastic membrane, the gas pressure will build up causing a pressure difference between the pressure in the inoculum well and the rest of the chamber. This will force the rupturable membrane into contact with the needle point ultimately resulting in the rupture of the membrane. Due to the elastic properties of the membrane it will contract to the well sides leaving a free light path through the inoculum well to the photocell. Electrolysis of the medium will be interrupted when the current flowing through the medium exceeds the current carrying capability of the fuse. With the blowing of the fuse, the voltage is removed from the electrodes and the electrolysis action ceases. The gas evolved by electrolysis will rapidly redissolve, and produce no undesirable denaturation or other deleterious effects in the medium. A wide variety of fuses are commercially available. The current rating of the fuse used is determined by the strength of the electrolytic solution, and the pressure build-up required to cause the rupture of the membrane.

It will be understood that various changes in the details, materials, steps and arrangements of parts, which have been herein described and illustrated in order to explain the nature of the invention, may be made by those skilled in the art within the principle and scope of this invention as expressed in the appended claims.

What is claimed is:

1. A biocell test device comprising: a light permeable hermetically sealed chamber for containing an aqueous electrolytic solution; light permeable enclosure means contained within said chamber for containing an inoculum of resting cells; a rupturable elastic membrane forming a part of said enclosure; electrolytic means for electrolysis of said electrolytic solution; piercing means for rupturing said membrane responsive to said electrolysis of said electrolytic solution; electric disconnecting means for interrupting said electrolysis; light emitting means exterior to said chamber and positioned so as to traverse said enclosure; and light sensing means providing an output potential responsive to light traversing said chamber and enclosure; said light sensing means connected to said electric disconnecting means and responsive to the light transmitting characteristics of said chamber and enclosure.

2. The device as set forth in claim 1 wherein said light sensing means is exterior to said chamber.

3. The device as set forth in claim 2 wherein said light sensing means comprises two photoresponsi-ve elements connected in electrical bridge relationship.

4. A biocell test device comprising: a light permeable hermetically sealed chamber for containing an aqueous electrolytic solution; light permeable enclosure means contained within said chamber for containing an inoculum of resting cells; a rupturable elastic membrane forming a part of said enclosure means; electrode means extending into said electrolytic solution for electrolysis of said solution; current responsive electric disconnect means connected in series with said electrode means; electric light emitting means exterior to said chamber and positioned so as to traverse said chamber and enclosure; piercing means contained in said enclosure means responsive to said electrolysis of said solution for rupturing said membrane; a first light sensing means exterior to said chamber responsive to light from said light emitting means traversing said chamber and enclosure; a second light sensing means exterior to said chamber responsive to said light emitting means; electrical resistive means: connecting means for connecting said first light sensing means, said second light sensing means, and said electrical resistive means in an electrical bridge circuit providing an output responsive to the light transmission characteristics of said chamber and enclosure; a source of electrical energy; and connecting means for connecting said source of electrical energy to said bridge circuit, said light emitting means and said series connected electrode and disconnect means.

References Cited UNITED STATES PATENTS 2,990,339 6/1961 Frank et al 103.5 3,322,956 5/1967 Shah 195103.5

ALVIN E. TANENHOLTZ, Primary Examiner. 

1. A BIOCELL TEST DEVICE COMPRISING: A LIGHT PERMEABLE HERMETICALLY SEALED CHAMBER FOR CONTAINING AN AQUEOUS ELECTROLYTIC SOLUTION; LIGHT PERMEABLE ENCLOSURE MEANS CONTAINED WITHIN SAID CHAMBER FOR CONTAINING AN INOCULUM OF RESTING CELLS; A RUPTURABLE ELASTIC MEMBRANE FORMING A PART OF SAID ENCLOSURE; ELECTROLYTIC MEANS FOR ELECTROLYSIS OF SAID ELECTROLYTIC SOLUTION; PIERCING MEANS FOR RUPTURING SAID MEMBRANE RESPONSIVE TO SAID ELECTROLYSIS OF SAID ELECTROLYTIC SOLUTION; ELECTRIC DISCONNECTING MEANS FOR INTERRUPTING SAID ELECTROLYSIS; LIGHT EMITTING MEANS EXTERIOR TO SAID CHAMBER AND POSITIONED SO AS TO TRAVERSE SAID ENCLOSURE; AND LIGHT SENSING MEANS PROVIDING AN OUTPUT POTENTIAL RESPONSIVE TO LIGHT TRAVERSING SAID CHAMBER AND ENCLOSURE; SAID LIGHT SENSING MEANS CONNECTED TO SAID ELECTRIC DISCONNECTING MEANS AND RESPONSIVE TO THE LIGHT TRANSMITTING CHARACTERISTICS FO SAID CHAMBER AND ENCLOSURE. 